Synthetic media for blood components

ABSTRACT

Synthetic media formulations are disclosed for use with blood preparations intended for in vivo use, including synthetic media formulations to be employed in conjunction with the photodecontamination of platelets.

The present application is a continuation of patent application Ser. No.09/240,067, filed Jan. 29, 1999, now U.S. Pat. No. 6,251,580, which is acontinuation of patent application Ser. No. 08/692,444, filed Aug. 5,1996, now U.S. Pat. No. 5,908,742, which is a continuation of patentapplication Ser. No. 08/338,039, filed Nov. 14, 1994, abandoned, whichis a continuation-in-part of patent application Ser. No. 08/072,485,filed Jun. 2, 1993, now U.S. Pat. No. 5,459,030 which is acontinuation-in-part of patent application Ser. No. 07/926,477, filedAug. 7, 1992, now, abandoned, which is a continuation-in-part of patentapplication Ser. No. 07/844,790, filed Mar. 2, 1992 and now U.S. Pat.No. 5,288,605 issued Feb. 22, 1994.

FIELD OF THE INVENTION

The invention generally relates to synthetic media for use with bloodpreparations intended for in vivo use, including synthetic media used inconjunction with the photodecontamination of platelets.

BACKGROUND

Whole blood collected from volunteer donors for transfusion recipientsis typically separated into its components: red blood cells, platelets,and plasma. Each of these fractions are individually stored and used totreat a multiplicity of specific conditions and disease states. Forexample, the red blood cell component is used to treat anemia, theconcentrated platelet component is used to control bleeding, and theplasma component is used frequently as a source of Clotting Factor VIIIfor the treatment of hemophilia.

Ideally, all blood cell preparations should be from freshly drawn bloodand then immediately transfused to the recipient. However, the logisticsof operating a blood donor center preclude this possibility in the vastmajority of cases. Transfusions are needed day and night and it isdifficult, if not impossible, to arrange for donor recruiting at unusualhours. Consequently, modern blood donor centers must use stored bloodproducts.

In the United States, blood storage procedures are subject to regulationby the government. The maximum storage periods for the blood componentscollected in these systems are specifically prescribed. For example,whole blood components collected in an “open” (i.e., non-sterile) systemmust, under governmental rules, be transfused within twenty-four hoursand in most cases within six to eight hours. By contrast, when wholeblood components are collected in a “closed” (i.e., sterile) system thered blood cells can be stored up to forty-two days (depending upon thetype of anticoagulant and storage medium used) and plasma may be frozenand stored for even longer periods.

While red cells are stored in the cold, Murphy and Gardner, New Eng. J.Med. 280:1094 (1969), demonstrated that platelets stored asplatelet-rich plasma (PRP) at 22° C. possessed a better in vivohalf-life than those stored at 4° C. Thus, more acceptable plateletconcentrates could be transfused after storage at room temperature.Until recently, the rules allowed for platelet concentrate storage atroom temperature for up to seven days (depending upon the type ofstorage container). However, it was recognized that the incidence ofbacterial growth and subsequent transfusion reactions in the recipientincreased to unacceptable levels with a seven day old plateletconcentrate. Platelet concentrates may now be stored for no more thanfive days.

One might believe that it is a relatively simple matter to keep theblood preparation sterile during the manipulations needed to concentratethe platelets. After all, blood bags used for platelet concentratepreparation are in themselves sterile, as are the connected satellitebags. However, bacteria can be introduced by at least two differentmeans.

First, if the donor is experiencing a mild bacteremia, the blood will becontaminated, regardless of the collection or storage method. Adequatedonor histories and physicals will decrease but not eliminate thisproblem. See B. J. Grossman et al., Transfusion 31:500 (1991).

A second, more pervasive source of contamination is the venepuncture.Even when “sterile” methods of skin preparation are employed, it isextremely difficult to sterilize the crypts around the sweat glands andhair follicles. During venepuncture, this contaminated skin is often cutout in a small “core” by a sharp needle. This core can serve to “seed”the blood bag with bacteria that may grow and become a risk to therecipient.

Indeed, many patients requiring platelet transfusions lack host-defensemechanisms for normal clearing and destruction of bacteria because ofeither chemotherapy or basic hematologic disease. The growth of evenseemingly innocuous organisms in stored platelets can, upon transfusion,result in recipient reaction and death. See e.g., B. A. Myhre, JAMA244:1333 (1980) and J. M. Heal et al., Transfusion 27:2 (1987).

The reports assessing the extent of contamination in platelets havediffered in their methods, sample size, and bacterial detection schemes.D. H. Buchholz et al., Transfusion 13:268 (1973) reported an overalllevel of platelet contamination of 2.4% when a large (>1000 bags) samplewas examined and extensive measures were taken for bacterial culturing.While some units were heavily contaminated after just 24 hours ofstorage, the incidence as a whole varied according to the age of theconcentrate and increased with the widespread practice of poolingindividual units; over 30% of pools were contaminated at 3 days. Seealso D. H. Buccholz et al., New Eng. J. Med. 285:429 (1971). While otherclinicians suggest lower numbers, recent studies indicate that septicplatelet transfusions are significantly underreported. See e.g., J. F.Morrow et al., JAMA 266:555 (1991).

Pre-culturing platelets is not a solution to the bacterial contaminationproblem. The culture assay takes 48 hours to detect growth. Holdingplatelet units for an additional two days to await the results of theassay would create, ironically, a smaller margin of safety. See Table 2in J. F. Morrow et al., JAMA 266:555 (1991). While heavily contaminatedunits would be detected at the outset, lightly contaminated units wouldbe allowed to grow for two days. Older and potentially more contaminatedunits would end up being transfused.

Washing the blood cells (e.g., with saline) or filtering the bacteriaare also not practical solutions. These techniques are time consumingand inefficient, as they can reduce the number of viable blood cellsavailable for transfusion. Most importantly, they typically involve an“entry” into the storage system. Once an entry is made in a previouslyclosed system, the system is considered “opened,” and transfusion mustoccur quickly, regardless of the manner in which the blood was collectedand processed in the first place.

Finally, antibiotics are not a reasonable solution. Contamination occursfrom a wide spectrum of organisms. Antibiotics would be needed to coverthis spectrum. Many recipients are allergic to antibiotics. In addition,there is an every increasing array of drug-resistant strains of bacteriathat would not be inactivated.

In sum, there is a need for a means of inactivating organisms from bloodcomponents prior to storage and transfusion in a way that lends itselfto use in a closed system. This approach must be able to handle avariety of organisms without harm to the blood product or thetransfusion recipient.

SUMMARY OF THE INVENTION

The invention generally relates to synthetic media for use with bloodpreparations intended for in vivo use, including synthetic media used inconjunction with the photodecontamination of platelets. By the term“synthetic media” the present invention intends to indicate aqueoussolutions (e.g., phosphate buffered, aqueous salt solutions) other thanthose found as natural fluids (e.g., plasma, serum, etc.). However, itis not intended that such synthetic media be used without the benefit ofnatural fluids. Indeed, in preferred embodiments, mixtures of syntheticsalt solutions and natural fluids are contemplated.

The present invention contemplates that the activating means comprises aphotoactivation device capable of emitting a given intensity of aspectrum of electromagnetic radiation comprising wavelengths between 180nm and 400 nm, and in particular, between 320 nm and 380 nm. It ispreferred that the intensity is less than 25 mW/cm² (e.g. between 10 and20 mW/cm²) and that the mixture is exposed to this intensity for betweenone and twenty minutes (e.g. ten minutes).

By the phrase “the maximum solubility of psoralen in water” the presentinvention intends a concentration derived experimentally in an aqueoussolution in the absence of organic solvents (e.g., DMSO, ethanol, etc.)at approximately room temperature. Concentrations exceeding this levelare detected by the presence of precipitate, which is undesirable forintravenous infusion.

A saturated solution of 8-methoxypsoralen can be made by simplydissolving the compound (over a number of hours at room temperature) indistilled water until precipitate is apparent. If the solution is simplycentrifuged, the supernatant can have a concentration of over 50 μg/ml.On the other hand, if the solution is filtered (e.g., glass wool), theconcentration of 8-methoxypsoralen has been found to be under 50 μg/ml.If, instead of centrifuging or filtering, the saturated solution isdialyzed against distilled water (over a number of days at roomtemperature), the compound is found to have a maximum solubility ofapproximately 39 μg/ml. It has been found that, when placed in acontainer (not glass) to shield the compound from the light, a 0.9% NaClsolution of 8-methoxypsoralen at a concentration of 30 μg/ml is stable.

When weighing components, some experimental variability is expected. Thepresent invention employs the term “approximately” to reflect thisvariability. This variability is typically plus or minus 5% and usuallyless than 10%.

In one embodiment, the present invention contemplates a syntheticplatelet storage media, comprising an aqueous solution of: 45-120 mMsodium chloride; 5-15 mM sodium citrate; 20-40 mM sodium acetate; and20-30 mM sodium phosphate. In a preferred embodiment, the aqueoussolution comprises: approximately 86 mM sodium chloride; approximately10 mM sodium citrate; approximately 30 mM sodium acetate; andapproximately 26 mM sodium phosphate. The solution has a pH ofapproximately pH 7.2 and an osmolarity of approximately 300 mOsm/Kg. Bynot containing glucose or magnesium, the media is readily autoclaved.

DESCRIPTION OF THE FIGURES

FIG. 1 is a perspective view of one embodiment of the device of thepresent invention.

FIG. 2 is a cross-sectional view of the device shown in FIG. 1 along thelines of 2—2.

FIG. 3 is a cross-sectional view of the device shown in FIG. 1 along thelines of 3—3.

FIG. 4 is a cross-sectional view of the device shown in FIG. 1 along thelines of 4—4.

FIG. 5 schematically shows the decontamination approach of the presentinvention applied specifically to blood products.

FIG. 6 is a graph showing the photoaddition of 8-methoxypsoralen tonucleic acid.

FIG. 7 is a graph showing the degradation of 8-methoxypsoralen (8-MOP)compared to that of 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT), asmeasured by HPLC.

FIG. 8 is a graph showing the inactivation of gram negative bacteria.

FIG. 9A schematically shows the standard blood product separationapproach used presently in blood banks.

FIG. 9B schematically shows an embodiment of the present inventionwhereby synthetic media is introduced to platelet concentrate preparedas in FIG. 9A.

FIG. 9C schematically shows one embodiment of the decontaminationapproach of the present invention applied specifically to plateletconcentrate diluted with synthetic media as in FIG. 9B.

DESCRIPTION OF THE INVENTION

The invention generally relates to synthetic media for use with bloodpreparations intended for in vivo use, including synthetic media used inconjunction with the photodecontamination of platelets.

As noted previously, whole blood is collected and typically separatedinto red blood cells, platelets, and plasma. Each of these fractions areindividually stored under specific conditions prior to in vivo use. Inmany cases, the extent of contamination is related to the storage timebecause of growth. A process that inactivated microorganisms at the timeof blood collection would be expected to prevent growth during storage.

In one embodiment, the present invention contemplates inactivating bloodproducts after separation but before storage. In this embodiment, anucleic acid binding compound is selectively employed to treatcontamination by microorganisms.

In one embodiment, the nucleic acid binding compound is selected fromthe group comprising furocoumarins. In a preferred embodiment, thefurocoumarin is a psoralen that is activated by a photoactivationdevice.

Psoralens are tricyclic compounds formed by the linear fusion of a furanring with a coumarin. Psoralens can intercalate between the base pairsof double-stranded nucleic acids, forming covalent adducts to pyrimidinebases upon absorption of longwave ultraviolet light (UVA). G. D. Ciminoet al., Ann. Rev. Biochem. 54:1151 (1985); and Hearst et al., Quart.Rev. Biophys. 17:1 (1984). If there is a second pyrimidine adjacent to apsoralen-pyrimidine monoadduct and on the opposite strand, absorption ofa second photon can lead to formation of a diadduct which functions asan interstrand crosslink. S. T. Isaacs et al., Biochemistry 16:1058(1977); S. T. Isaacs et al., Trends in Photobiology (Plenum)pp. 279-294(1982); J. Tessman et al., Biochem. 24:1669 (1985); and Hearst et al.,U.S. Pat. Nos. 4,124,589, 4,169,204, and 4,196,281, hereby incorporatedby reference.

Psoralens have been shown to inactivate viruses in some blood products.See H. J. Alter et al., The Lancet (ii:1446) (1988); L. Lin et al.,Blood 74:517 (1989); and G. P. Wiesehahn et al., U.S. Pat. Nos.4,727,027 and 4,748,120, hereby incorporated by reference, describe theuse of a combination of 8-methoxypsoralen (8-MOP) and irradiation. Theyshow that 300 μg/ml of 8-MOP together with one hour or more ofirradiation with ultraviolet light can effectively inactivate viruses.However, these treatment conditions cause harm to the blood productbecause of energy transfer. Their approach is only feasible if thedamage to cells is specifically suppressed by limiting the concentrationof molecular oxygen, a difficult and expensive process.

The inactivation method of the present invention provides a method ofinactivating single cell and multicellular organisms, and in particular,bacteria, fungi, mycoplasma and protozoa. In addition, the presentinvention contemplates inactivation of viruses (e.g. HIV virus). Incontrast to previous approaches, the method of the present inventiondoes not cause harm to the blood product. There is no significant damageto cells and, therefore, no need to limit the concentration of molecularoxygen.

The present invention contemplates using much lower concentrations ofnucleic acid binding compounds than previously employed. For example,the present invention contemplates using 8-MOP at concentrations of 30ug/ml or less. Indeed, a preferred concentration of 8-MOP for bacterialdecontamination in platelet concentrates is 3 μg/ml or less, i.e., a onehundred-fold lower concentration than employed by G. P. Wiesehahn etal., supra. Because lower concentrations are employed, solvents likeDMSO (used to increase the solubility of 8-MOP) are unnecessary.

The present invention, furthermore, contemplates using much lower dosesof irradiation than previously described. This is accomplished withlower intensity irradiation sources, with wavelength cutoff filters (seebelow), and/or shorter irradiation times. In a preferred embodiment, thetime of irradiation is variable and controlled from 1 second to 99minutes, in one second increments.

In one embodiment, the device of the present invention is mounted on anagitator, giving horizontal unidirectional and sinusoidal motion ofvariable frequency and amplitude. In another embodiment, heat from thelamps, ballasts and other sources is blocked from the bags.

While it is not intended that the present invention be limited by thetheory of inactivation, the use of lower compound concentrations andirradiation doses comes from an understanding that, where the presentinvention is applied to the decontamination of a single cell ormulticellular organism, a lower level of nucleic acid binding willachieve inactivation. In addition, it is recognized that it is notessential that inactivation be complete. That is to say, partialinactivation will be adequate as long as the viable portion is unable,within the storage period, to grow to levels sufficient to causedisease.

To appreciate that, in any given case, an inactivation method may or maynot achieve complete inactivation, it is useful to consider a specificexample. A bacterial culture is said to be sterilized if an aliquot ofthe culture, when transferred to a fresh culture plate and permitted togrow, is undetectable after a certain time period. The time period andthe growth conditions (e.g., temperature) define an “amplificationfactor”. This amplification factor along with the limitations of thedetection method (e.g., visual inspection of the culture plate for theappearance of a bacterial colony) define the sensitivity of theinactivation method. A minimal number of viable bacteria must be appliedto the plate for a signal to be detectable. With the optimum detectionmethod, this minimal number is 1 bacterial cell. With a suboptimaldetection method, the minimal number of bacterial cells applied so thata signal is observed may be much greater than 1. The detection methoddetermines a “threshold” below which the method appears to be completelyeffective (and above which the method is, in fact, only partiallyeffective).

This interplay between the amplification factor of an assay and thethreshold that the detection method defines, can be illustrated. Forexample, bacterial cells can be applied to a plate; the detection methodis arbitrarily chosen to be visual inspection. Assume the growthconditions and time are such that an overall amplification of 10⁴ hasoccurred. The detectable signal will be proportional to the number ofbacterial cells actually present after amplification. For calculationpurposes, the detection threshold is taken to be 10⁶ cells; if fewerthan 10⁶ cells are present after amplification, no cell colonies arevisually detectable and the inactivation method will appear effective.Given the amplification factor of 10⁴ and a detection threshold of 10⁶,the sensitivity limit would be 100 bacterial cells; if less than 100viable bacterial cells were present in the original aliquot of thebacterial culture after the sterilization method is performed, theculture would still appear to be sterilized.

Such a situation is common for bacterial growth assays. The sensitivityof the assay is such that viable bacterial cells are present but theassay is unable to detect them. This may explain, at least in part, thevariability in results obtained by researchers attempting to determinethe extent of bacterial contamination of blood products. See D. H.Buchholz, et al., Transfusion 13:268 (1973), wherein such variability isdiscussed.

It should be noted that, in many countries, contamination of bloodproducts by cellular organisms is more pervasive and, therefore, moreserious than viral contamination. For example, in South America, themost important blood-borne organism is T. cruzi, which is the etiologicagent of Chagas disease. Approximately 16-18 million people are infectedin the Americas (including 11% of the population of Chile). It iscontemplated that the decontamination method of the present invention iswell-suited for inactivation of this protozoa.

The present invention contemplates specific synthetic media formulationsfor use with blood preparations. The formulations of the presentinvention are particularly useful for platelet storage. Theseformulations are also useful when employed in conjunction with thephotodecontamination of platelets.

While synthetic media have not previously been employed in conjunctionwith photodecontamination, synthetic media formulations have beendescribed for platelet storage. For example, U.S. Pat. No. 4,828,976 toMurphy stresses the importance of having a synthetic blood plateletstorage medium that is essentially free of glucose and calcium. Thespecification notes that glucose consumption and lactate productioncontinue as long as glucose is present at the concentration above 1.5 mM(Col. 12, line 13). Lactate production is thought to be the cause ofloss of pH stability and consequent impairment of platelet function. Seealso U.S. Pat. No. 4,925,665 and U.S. Pat. No. 4,992,363.

U.S. Pat. No. 4,447,415 to Rock describes a synthetic medium consistingessentially of: a balanced, physiologically compatible, saline solution;an anticoagulant; and one or more additives to enhance stability of theplatelets selected from: (a) nutrients to improve the storage life ofthe platelets; (b) reversible inhibitors for platelet activation; (c)substances to raise cyclic adenosine monophosphate levels which havereversible effects on platelets; and (d) buffering agents which arephysiologically compatible. The specification stresses the need toremove plasma. The specification indicates that this is achieved eitherby centrifugation and washing, or by “extraction” (see Examples 1 and 2of the '415 patent). See also U.S. (Reissue) Pat. No. 32,874.

U.S. Pat. No. 4,704,352 to Miripol discloses a medium which containseither magnesium L-ascorbate-2-phosphate or calciumL-ascorbate-2-phosphate. U.S. Pat. No. 4,695,460 to Holme describe mediathat contain sodium bicarbonate U.S. Pat. No. 4,390,619 toHarmening-Pittiglio discloses a media capable of supporting plateletmetabolism, containing a water-insoluble polymer containing releasablephosphate or bicarbonate ions.

In contrast to these previous attempts, the formulations of the presentinvention: i) utilize residual plasma; and ii) avoid bicarbonate. Withrespect to plasma, the present invention contemplates that a standardplasma expression method (e.g., such as currently used in most bloodbanks) will be used, such that platelets are suspended in a residualplasma concentration between 8 and 25% by volume, and more commonly 12to 20%. Bicarbonate has been found to be extremely difficult to controlin a system employing gas permeable blood bags. The present inventiontherefore contemplates a phosphate buffer to control the pH of the bloodpreparation.

The present invention contemplates devices and methods forphotoactivation and specifically, for activation of photoreactivenucleic acid binding compounds. The present invention contemplatesdevices having an inexpensive source of electromagnetic radiation thatis integrated into a unit. In general, the present inventioncontemplates a photoactivation device for treating photoreactivecompounds, comprising: a) means for providing appropriate wavelengths ofelectromagnetic radiation to cause activation of at least onephotoreactive compound; b) means for supporting a plurality of bloodproducts in a fixed relationship with the radiation providing meansduring activation; and c) means for maintaining the temperature of theblood products within a desired temperature range during activation. Thepresent invention also contemplates methods, comprising: a) supporting aplurality of blood product containers, containing one or morephotoreactive compounds, in a fixed relationship with a fluorescentsource of electromagnetic radiation; b) irradiating the plurality ofblood products simultaneously with said electromagnetic radiation tocause activation of at least one photoreactive compound; and c)maintaining the temperature of the blood products within a desiredtemperature range during activation.

The major features of one embodiment of the device of the presentinvention involve: A) an inexpensive source of ultraviolet radiation ina fixed relationship with the means for supporting the sample vessels;B) rapid photoactivation; C) large sample processing; D) temperaturecontrol of the irradiated samples; and E) inherent safety.

A. Electromagnetic Radiation Source

A preferred photoactivation device of the present invention has aninexpensive source of ultraviolet radiation in a fixed relationship withthe means for supporting the sample vessels. Ultraviolet radiation is aform of energy that occupies a portion of the electromagnetic radiationspectrum (the electromagnetic radiation spectrum ranges from cosmic raysto radio waves). Ultraviolet radiation can come from many natural andartificial sources. Depending on the source of ultraviolet radiation, itmay be accompanied by other (non-ultraviolet) types of electromagneticradiation (e.g., visible light).

Particular types of ultraviolet radiation are herein described in termsof wavelength. Wavelength is herein described in terms of nanometers(“nm”; 10⁻⁹ meters). For purposes herein, ultraviolet radiation extendsfrom approximately 180 nm to 400 nm. When a radiation source, by virtueof filters or other means, does not allow radiation below a particularwavelength (e.g., 320 nm), it is said to have a low end “cutoff” at thatwavelength (e.g., “a wavelength cutoff at 300 nanometers”). Similarly,when a radiation source allows only radiation below a particularwavelength (e.g., 360 nm), it is said to have a high end “cutoff” atthat wavelength (e.g., “a wavelength cutoff at 360 nanometers”).

For any photochemical reaction it is desired to eliminate or leastminimize any deleterious side reactions. Some of these side reactionscan be caused by the excitation of endogenous chromophores that may bepresent during the photochemical activation procedure. In a system whereonly nucleic acid and psoralen are present, the endogenous chromophoresare the nucleic acid bases themselves. Restricting the activationprocess to wavelengths greater than 320 nm minimizes direct nucleic aciddamage since there is very little absorption by nucleic acids above 313nm.

In blood products, the nucleic acid is typically present together withadditional biological chromophores. If the biological fluid is justprotein, the 320 nm cutoff will be adequate for minimizing sidereactions (aromatic amino acids do not absorb above 320 nm). If thebiological fluid includes cells and/or cellular constituents, there willbe many other chromophores, including hemes and flavins.

Hemes are abundant in blood products where they arise from the lysis ofred cells. Flavins, like hemes, are required for metabolic respiration.Both of these endogenous chromophores will cause damage to cells ifexcited by photoirradiation.

Hemes have three principle absorption bands: two are in the red regionof the visible spectrum; the other is centered about 400 nm. Flavinshave two principle absorption peaks: one at 450 nm and the other at 370nm.

In view of the presence of these endogenous chromophores in bloodproducts, it is intended that the device of the present invention bedesigned to allow for irradiation within a small range of specific anddesirable wavelengths, and thus avoid damage to cells caused by energytransfer. The preferred range of desirable wavelengths is between 320and 350 nm.

Some selectivity can be achieved by choice of commercial irradiationsources. For example, while typical fluorescent tubes emit wavelengthsranging from 300 nm to above 400 nm (with a broad peak centered around360 nm), BLB type fluorescent lamps are designed to remove wavelengthsabove 400 nm. This, however, only provides an upper end cutoff.

In a preferred embodiment, the device of the present invention comprisesan additional filtering means. In one embodiment, the filtering meanscomprises a glass cut-off filter, such as a piece of Cobalt glass. Inanother embodiment, the filtering means comprises a liquid filtersolution that transmit only a specific region of the electromagneticspectrum, such as an aqueous solution of Co(No₃)₂. This salt solutionyields a transmission window of 320-400 nm. In a preferred embodiment,the aqueous solution of Co(No₃)₂ is used in combination with NiSO₄ toremove the 365 nm component of the emission spectrum of the fluorescentor arc source employed. The Co—Ni solution preserves its initialtransmission remarkably well even after tens of hours of exposure to thedirect light of high energy sources.

It is not intended that the present invention be limited by theparticular filter employed. Several inorganic salts and glasses satisfythe necessary requirements. For example, cupric sulfate is a most usefulgeneral filter for removing the infra-red, when only the ultraviolet isto be isolated. Its stability in intense sources is quite good. Othersalts are known to one skilled in the art. Aperture or reflector lampsmay also be used to achieve specific wavelengths and intensities.

When ultraviolet radiation is herein described in terms of irradiance,it is expressed in terms of intensity flux (milliwatts per squarecentimeter or “mW cm⁻²”). “Output” is herein defined to encompass boththe emission of radiation (yes or no; on or off) as well as the level ofirradiance. In a preferred embodiment, intensity is monitored at 4locations: 2 for each side of the plane of irradiation.

A preferred source of ultraviolet radiation is a fluorescent source.Fluorescence is a special case of luminescence. Luminescence involvesthe absorption of electromagnetic radiation by a substance and theconversion of the energy into radiation of a different wavelength. Withfluorescence, the substance that is excited by the electromagneticradiation returns to its ground state by emitting a quantum ofelectromagnetic radiation. While fluorescent sources have heretoforebeen thought to be of too low intensity to be useful forphotoactivation, in one embodiment the present invention employsfluorescent sources to achieve results thus far achievable on onlyexpensive equipment.

As used here, fixed relationship is defined as comprising a fixeddistance and geometry between the sample and the light source during thesample irradiation. Distance relates to the distance between the sourceand the sample as it is supported. It is known that light intensity froma point source is inversely related to the square of the distance fromthe point source. Thus, small changes in the distance from the sourcecan have a drastic impact on intensity. Since changes in intensity canimpact photoactivation results, changes in distance are avoided in thedevices of the present invention. This provides reproducibility andrepeatability.

Geometry relates to the positioning of the light source. For example, itcan be imagined that light sources could be placed around the sampleholder in many ways (on the sides, on the bottom, in a circle, etc.).The geometry used in a preferred embodiment of the present inventionallows for uniform light exposure of appropriate intensity for rapidphotoactivation. The geometry of a preferred device of the presentinvention involves multiple sources of linear lamps as opposed to singlepoint sources. In addition, there are several reflective surfaces andseveral absorptive surfaces. Because of this complicated geometry,changes in the location or number of the lamps relative to the positionof the samples to be irradiated are to be avoided in that such changeswill result in intensity changes.

B. Rapid Photoactivation

The light source of the preferred embodiment of the present inventionallows for rapid photoactivation. The intensity characteristics of theirradiation device have been selected to be convenient with theanticipation that many sets of multiple samples may need to beprocessed. With this anticipation, a fifteen minute exposure time orless is a practical goal.

In designing the devices of the present invention, relative position ofthe elements of the preferred device have been optimized to allow forfifteen minutes of irradiation time, so that, when measured for thewavelengths between 320 and 350 nanometers, an intensity flux greaterthan approximately 1 mW cm⁻² is provided to the sample vessels. In apreferred embodiment, the device irradiates both sides of the bag.

C. Processing of Large Numbers of Samples

As noted, another important feature of the photoactivation devices ofthe present invention is that they provide for the processing of largenumbers of samples. In this regard, one element of the devices of thepresent invention is a means for supporting a plurality of bloodproducts, and in particular, blood bags. In the preferred embodiment ofthe present invention the supporting means comprises glass platesbetween two banks of lights with a capacity of six 50 ml bags(equivalent to Dupont Stericell bag) plus connectors and tubing, at onetime. By accepting commonly used commercially available blood bags, thedevice of the present invention allows for convenient processing oflarge numbers of samples.

D. Temperature Control

As noted, one of the important features of the photoactivation devicesof the present invention is temperature control. Temperature control isimportant because the temperature of the sample in the sample at thetime of exposure to light can dramatically impact the results. Forexample, conditions that promote secondary structure in nucleic acidsalso enhance the affinity constants of many psoralen derivatives fornucleic acids. Hyde and Hearst, Biochemistry, 17, 1251 (1978). Theseconditions are a mix of both solvent composition and temperature. Withsingle stranded 5S ribosomal RNA, irradiation at low temperaturesenhances the covalent addition of HMT to 5S rRNA by two fold at 4° C.compared to 20° C. Thompson et al., J. Mol. Biol. 147:417 (1981). Evenfurther temperature induced enhancements of psoralen binding have beenreported with synthetic polynucleotides. Thompson et al., Biochemistry21:1363 (1982).

With respect to bacteria, it should be noted that repair of crosslinksoccurs during irradiation. However, where a lower temperature isemployed during irradiation, the bacterial repair process is suppressed.Thus, a 15° C. irradiation has a significant effect on the level ofinactivation that is observed.

E. Inherent Safety

Ultraviolet radiation can cause severe burns. Depending on the nature ofthe exposure, it may also be carcinogenic. The light source of apreferred embodiment of the present invention is shielded from the user.This is in contrast to the commercial hand-held ultraviolet sources aswell as the large, high intensity sources. In a preferred embodiment,the irradiation source is contained within a housing made of materialthat obstructs the transmission of radiant energy (i.e., an opaquehousing). No irradiation is allowed to pass to the user. This allows forinherent safety for the user.

EXPERIMENTAL

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); gm (grams); mg (milligrams); μg (micrograms); L (liters);ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters);μm (micrometers); nm (nanometers); ° C. (degrees Centigrade); HPLC (HighPressure Liquid Chromatography).

In some of the examples below, phosphate buffered synthetic media isformulated for platelet storage and treatment. One embodiment is made asfollows:

Ingredients Amounts (grams/liter) Na Acetate.3H₂O 4.08 Citrate.2H₂O 2.94NaH₂PO₄.H₂O 0.858 Na₂HPO₄ 2.81 NaCl 5.02 pH adjustment to pH 7.2 (withHCl)

Distilled water is added to make 1 liter, the solution is mixed, sterilefiltered (0.2 micron filter) and refrigerated.

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

EXAMPLE 1

As noted above, the present invention contemplates devices and methodsfor the activation of photoreactive nucleic acid binding compounds. Inthis example, a photoactivation device is described for decontaminatingblood products according to the method of the present invention. Thisdevice comprises: a) means for providing appropriate wavelengths ofelectromagnetic radiation to cause activation of at least onephotoreactive compound; b) means for supporting a plurality of bloodproducts in a fixed relationship with the radiation providing meansduring activation; and c) means for maintaining the temperature of theblood products within a desired temperature range during activation.

FIG. 1 is a perspective view of one embodiment of the device integratingthe above-named features. The figure shows an opaque housing (100) witha portion of it removed, containing an array of bulbs (101) above andbelow a plurality of representative blood product containing means (102)placed between plate assemblies (103, 104). The plate assemblies (103,104) are described more fully, subsequently.

The bulbs (101), which are connectable to a power source (not shown),serve as a source of electromagnetic radiation. While not limited to theparticular bulb type, the embodiment is configured to accept an industrystandard, dual bipin lamp.

The housing (101) can be opened via a latch (105) so that the bloodproduct can be placed appropriately. As shown in FIG. 1, the housing(100), when closed, completely contains the irradiation from the bulbs(101). During irradiation, the user can confirm that the device isoperating by looking through a safety viewport (106) which does notallow transmission of ultraviolet light to the user.

The housing (100) also serves as a mount for several electroniccomponents on a control board (107), including, by way of example, amain power switch, a count down timer, and an hour meter. Forconvenience, the power switch can be wired to the count down timer whichin turn is wired in parallel to an hour meter and to the source of theelectromagnetic radiation. The count down timer permits a user to presetthe irradiation time to a desired level of exposure. The hour metermaintains a record of the total number of radiation hours that areprovided by the source of electromagnetic radiation. This featurepermits the bulbs (101) to be monitored and changed before their outputdiminishes below a minimum level necessary for rapid photoactivation.

FIG. 2 is a cross-sectional view of the device shown in FIG. 1 along thelines of 2—2. FIG. 2 shows the arrangement of the bulbs (101) with thehousing (100) opened. A reflector (108A, 108B) completely surrounds eacharray of bulbs (101). Blood product containing means (102) are placedbetween upper (103) and lower (104) plate assemblies. Each plateassembly is comprised of an upper (103A, 104A) and lower (103B, 104B)plates. The plate assemblies (103, 104) are connected via a hinge (109)which is designed to accommodate the space created by the blood productcontaining means (102). The upper plate assembly (103) is brought torest gently on top of the blood product containing means (102) supportedby the lower plate (104B) of the lower plate assembly (104).

Detectors (110A, 110B, 110C, 110D) may be conveniently placed betweenthe plates (103A, 103B, 104A, 104B) of the plate assemblies (103, 104).They can be wired to a printed circuit board (111) which in turn iswired to the control board (107).

FIG. 3 is a cross-sectional view of the device shown in FIG. 1 along thelines of 3—3. Six blood product containing means (102) (e.g., Teflon™platelet unit bags) are placed in a fix relationship above an array ofbulbs (101). The temperature of the blood product can be controlled viaa fan (112) alone or, more preferably, by employing a heat exchanger(113) having cooling inlet (114) and outlet (115) ports connected to acooling source (not shown).

FIG. 4 is a cross-sectional view of the device shown in FIG. 1 along thelines of 4—4. FIG. 4 more clearly shows the temperature control approachof a preferred embodiment of the device. Upper plate assembly plates(103A, 103B) and lower plate assembly plates (104A, 104B) each create atemperature control chamber (103C, 104C), respectively. The fan (112)can circulate air within and between the chambers (103C, 104C). When theheat exchanger (113) is employed, the circulating air is cooled andpassed between the plates (103A, 103B, 104A, 104B).

EXAMPLE 2

FIG. 5 shows an embodiment wherein platelets are treated by the methodof the present invention. Following fractionation, platelets aretransferred to a bag containing a nucleic acid binding compound (shownin FIG. 5 as a shaded bag). This bag, which has transmission propertiesand other characteristics suited for the present invention, is thenplaced in an irradiation device (such as that described in Example 1,above) and is irradiated. The free compound may be collected or“captured” as desired by a capture device. In such a case, the bag wouldcontain only compound that is contained in cells; the bag would have nofree compound (this bag is indicated in FIG. 5 as unshaded).

In one capture approach, a bag comprised of a psoralen-binding polymeris employed to capture the compound. The Cutter CLX bag has been foundto have this property.

EXAMPLE 3

In this example, the decontamination methods of the present inventionare applied to inactivate Klebsiella pneumoniae, which is known to beamong the organisms associated with bacteremia. See generally, Infect.Control 5:343 (1984). The particular isolate in this case was obtainedfollowing a platelet transfusion where the recipient immediately wentinto shock and later died. The platelet bag was obtained and cultured,and the organism was identified and serotyped.

For the experiment, the strain was kept at ambient temperature andinoculated onto either heart infusion agar (HIA) or heart infusion agarcontaining 5% (v/v) sheep blood (BAP) by swabbing each plate forconfluency via a sterile applicator swab. Cultures were then incubatedunder static conditions for 16-18 h at 35° C. At the end of theincubation period, cultures were removed and suspended in phosphatebuffered saline (PBS;pH 7.2-7.4) and spectrophotometrically standardizedto 1.0 at an OD₆₁₀ using a Spectronic 501 or 601 spectrophotometer(Bausch and Lomb). After standardization, suspensions were diluted 1:10in PBS to achieve an ca. 10⁸ CFU/ml concentration. This standardizedsuspension is then split to use an aliquot for the inactivation study,while another portion was plated in duplicate 10-fold serial dilutionsonto HIA (or BAP) to ensure appropriate concentrations of the organism.

To assess inactivation of the organism, two ABO compatible freshlyoutdated human platelet concentrate units were obtained from the BloodBank of Alameda—Contra Costa Medical Association. They were pooled andredivided into two bags. One bag was infused with the bacteriapreparation. The platelets in the second unit were pelleted at 4000×gfor 6 minutes and then resuspended in a medium containing 85% saline and15% plasma. Bacteria was added after platelets were well resuspended.

3 ml aliquots of bacteria containing platelet concentrate weretransferred to a Teflon™ minibag (American Fluoroseal Corporation,Silver Spring, Md.) and received specified amounts of 8-MOP and UVAirradiation, except for the controls, which were irradiated withoutpsoralen, or received no treatment. Temperature was maintained at 25° C.during irradiation in the irradiation device described above which isequipped with an air cooling mechanism.

F24T 12-BL-HO fluorescent lamps were used. These are high output “blacklight” tubes (engineered to emit specific wavelengths by means of aninternal phosphor coating) 24 inches in length. Total intensity is lessthan 25 mw/cm² and typically between 15 and 20 mw/cm².

Following irradiation, bacteria were quantified by plating 0.1 ml ofserial 10-fold dilutions in broth onto 100 mm petri dishes containingBHI agar. After 24 hr incubation at 35° C., colonies were counted andbacterial concentration was calculated on a per ml basis. The results(FIG. 8) show that while only 1.2 logs of Klebsiella were killed by 5μg/ml 8-MOP in five minutes of UVA irradiation in 100% plasma, more than6.5 logs were killed under the same conditions in 15% plasma and 85%saline.

While not limited to any theory as to the mechanism by which thisimprovement in-decontamination efficiency was achieved, it would appearthat these results indicate that either that the optical properties ofthe synthetic medium are better, or the lower protein concentrationallows for a higher concentration of free 8-MOP. In the lattersituation, the 8-MOP would be more available for decontamination.

EXAMPLE 4

Artuc and co-workers examined the solubility of 8-MOP in human andbovine serum proteins. They showed that, when working with 8-MOPconcentrations ranging from 100 to 1000 ng/ml in serum, 75% to 80% ofthe 8-MOP was bound to albumin. M. Artuc et al., Brit. J. Derm. 101:669(1979).

In this example, the binding of 8-MOP to Calf Thymus DNA is comparedusing plasma and a protein free media in order to validate theefficiency of psoralen-nucleic interactions under the decontaminationmethods of the present invention. Although this measurement usedeukaryotic nucleic acid rather than bacterial nucleic acid, it is auseful indicator of the degree of adduct formation for bacteria.

³H-8-MOP was prepared to a concentration of 115 μg/ml in ethanol at aspecific activity of 4.7×10⁶ CPM/microgram (hereinafter “8-MOP stock”).Thereafter 130.5 or 22 μl of 8-MOP stock (2 each) for samples containingDNA (“+DNA”) and 52.2 or 8.7 μl for samples not containing DNA (“−DNA”)were dried down. To +DNA samples, 40 μl of DNA stock (7.7 mg/ml) wasadded as well as either 460 μl plasma (day old frozen) or 450 μlTris-EDTA (“TE”) buffer. To the latter was also added 10 μl 5M NaCl. For−DNA samples (i.e., the controls), 184 μl plasma and 16 μl water wasadded.

The samples were mildly vortexed for approximately one hour and thecounts were checked to confirm that the 8-MOP dissolved.

Each sample (100 ul) was irradiated on an HRI-100 (HRI Research Inc.,Concord, Calif.) at 25° C. for 0, 2, 4, 8, and 16 minutes. Samples werekept at 4° C. overnight after irradiation. Thereafter, the samples wereextracted. First, a phenol solution was prepared at pH 8 byequilibrating with 0.1M Tris pH 8. Each sample was then extracted with100 μl phenol. Each sample was centrifuged for 5 minutes to remove theaqueous phase to a new tube. A second extraction was performed with 100μl 1:1 phenol:chloroform. A final extraction was performed with 100 μlchloroform.

The final aqueous phase was precipitated by adding 50 μl NaCl adjustedto give a final concentration of NaCl of 0.2M and then adding 250 μlethanol. The samples were again centrifuged (10 minutes). Thesupernatant was removed and the pellets were dried. The pellets wereresuspended in 100 μl TE and reprecipitated. This was repeated for atotal of 3 precipitations. The final pellets were brought up in 600 μlwater and 100 ul was counted. Each sample was assayed for DNA bymeasuring absorbance (260 nm). 8-MOP levels were plotted as adducts per1000 base pairs (“8-MOP:kBP”).

The results (FIG. 6) show that plasma does significantly change theaddition kinetics of 8-MOP to DNA. Addition to nucleic acid is muchbetter in the protein free media.

The frequency of 8-MOP-DNA adduct formation in protein free mediapredicts a high multiplicity of modification of the bacterial genome.Furthermore, this type of biochemical measurement has the potential toprovide a means to monitor the efficiency of the photochemicalinactivation method.

EXAMPLE 5

Photoactivation of psoralens and isopsoralens may result in a variety ofphotoproducts. “Photoproduct” is best understood by considering thepossible reactions of photoreactive compound when exposed to activatingwavelengths of electromagnetic radiation. While not limited to anyprecise mechanism, it is believed that the reaction of photoreactivecompound in its ground state (“C”) with activating wavelengths ofelectromagnetic radiation creates a short-lived excited species (“C*”)):

C→C*

What happens next is largely a function of what potential reactants areavailable to the excited species. Since it is short-lived, a reaction ofthis species with nucleic acid (“NA”) is believed to only be possible ifnucleic acid is present at the time the excited species is generated.Thus, the reaction must, in operational terms, be in the presence ofactivating wavelengths of electromagnetic radiation, i.e., it is“photobinding”; it is not dark binding. The reaction can be depicted asfollows:

C*+NA→NA:C

The product of this reaction is hereinafter referred to as“Photoaddition Product” and is to be distinguished from “Photoproduct.”

With this reaction described, one can now consider the situation wherenucleic acid is not available for binding at the time the compound isexposed to activating wavelengths of electromagnetic radiation. Sincethe excited species is short-lived and has no nucleic acid to reactwith, the excited species may simply return to its ground state:

C*→C

On the other hand, the excited species may react with itself (i.e., aground state or excited species) to create a ground state complex(“C:C”). The product of these self-reactions where two compounds reactis referred to as “photodimer” or simply “dimer.” The self-reactions,however, are not limited to two compounds; a variety of multimers may beformed (trimers, etc.).

The excited species is not limited to reacting with itself. It may reactwith its environment, such as elements of the solvent (“E”) (e.g., ions,gases, etc.) to produce other products:

C*+E→E:C

It is this type of reaction that is believed to cause cellular damage(e.g.,, reaction with oxygen to create singlet oxygen species).Furthermore, it may simply internally rearrange (“isomerize”) to aground state derivative (“[”):

C*→[

Finally, the excited species may undergo other reactions than describedhere.

The present invention and the understanding of “photoproduct” does notdepend on which one (if any) of these reactions actually occurs.“Photoproduct”—whatever its nature—is deemed to exist if, following thereaction of a compound and activating wavelengths of electromagneticradiation, there is a resultant product formed that can interact withother components of the reaction environment.

With psoralens such as 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT),there are a number of resultant products produced when the HMT isexposed to activating wavelengths of electromagnetic radiation. Themajor resultant products of HMT are two cyclobutyl photodimers. In oneof the dimers, the two pyrone rings are linked in a cis-synconfiguration, while in the other dimer, the linkage occurs between thefuran end of one molecule and the pyrone end of the other, again withcis-syn configuration. A third resultant product of HMT is a monomericHMT photoisomer. In this isomer, the central ring oxygens assume a 1, 4instead of the normal 1, 3 orientation. While the two photodimers wouldnot be expected to have an intercalating activity due to geometricalconsiderations, the photoisomer remains planar, and accordingly, it iscontemplated that it has a positive intercalative association withdouble stranded nucleic acid and, thus, could be a mutagen.

In this example, the photochemical breakdown of 8-MOP is compared withAMT. The samples were analyzed by reverse phase HPLC using a RainenDynamax 300A column. Gradient elution was performed with 0.1M ammoniumacetate/acetonitrile (0-70% acetonitrile over 42 minutes). AMT elutes asa single peak at approximately 24 minutes under these conditions.Detection was by absorption at either 260 or 330 nm. The latterwavelength was used for the plasma containing samples.

Standard solutions of each compound were prepared at variousconcentrations. These solutions were then diluted 1:10 into water, then300 μl injected for analysis. All samples were monitored at 300 nm.Peaks were analyzed by measuring either peak height or peak area, thenconverted to a μg/ml value using the standard plot. Peak area wasdetermined by photocopying the trace, cutting out the copy of the peak,then weighing the resultant trace. The two methods gave essentially thesame result.

The results are shown in FIG. 7. Clearly, AMT degrades more quickly than8-MOP. It would, therefore, be expected to generate morephotoproducts—which eventually would end up in the transfusionrecipient. By contrast, it is not expected that 8-MOP generates asignificant amount of photoproducts. This is important when oneconsiders that the weight of authority has concluded that unactivated8-MOP is nonmutagenic.

EXAMPLE 6

FIG. 9 shows an embodiment wherein platelets are diluted with syntheticmedia by the method of the present invention. FIG. 9A schematicallyshows the standard blood product separation approach used presently inblood banks. Three bags are integrated by flexible tubing to create ablood transfer set (200) (e.g., commercially available from Baxter,Deerfield, Ill.). After blood is drawn into the first bag (201), theentire set is processed by centrifugation (e.g., Sorvall™ swing bucketcentrifuge, Dupont), resulting in packed red cells and platelet richplasma in the first bag (201). The plasma is expressed off of the firstbag (201) (e.g., using a Fenwall™ device for plasma expression), throughthe tubing and into the second bag (202). The first bag (201) is thendetached and the two bag set is centrifuged to create plateletconcentrate and platelet-poor plasma; the latter is expressed off of thesecond bag (202) into the third bag (203).

FIG. 9B schematically shows an embodiment of the present invention bywhich synthetic media is introduced to platelet concentrate prepared asin FIG. 9A. A two bag set (300) is sterile docked with the plateletconcentrate bag (202) (indicated as “P.C.”). Sterile docking iswell-known to the art. See e.g., U.S. Pat. No. 4,412,835 to D. W. C.Spencer, hereby incorporated by reference. See also U.S. Pat. Nos.4,157,723 and 4,265,280, hereby incorporated by reference. Steriledocking devices are commercially available (e.g., Terumo, Japan).

One of the bags (301) of the two bag set (300) contains a syntheticmedia formulation of the present invention (indicated as “STERILYTE”).In the second step shown in FIG. 9B, the platelet concentrate is mixedwith the synthetic media by transferring the platelet concentrate to thesynthetic media bag (301).

FIG. 9C schematically shows one embodiment of the decontaminationapproach of the present invention applied specifically to plateletconcentrate diluted with synthetic media as in FIG. 9B. In thisembodiment, platelets have been transferred to a synthetic media bag(301) containing a nucleic acid binding compound. This bag (301), whichhas UV light transmission properties and other characteristics suitedfor the present invention, is then placed in a device (such as thatdescribed in Example 1, above) and illuminated.

Following phototreatment, the platelets are transferred from thesynthetic media bag (301) into the storage bag (302) of the two bag set(300). The storage bag can be a commercially available storage bag(e.g., CLX bag from Cutter).

From the above, it should be evident that the present invention providessynthetic media formulations for use with blood preparations intendedfor storage and in vivo use. The formulations include synthetic mediaformulations to be employed in conjunction with the photodecontaminationof platelets.

We claim:
 1. A magnesium-free and glucose-free, synthetic plateletstorage media comprising an aqueous solution of: 45-120 mM sodiumchloride; 5-15 mM sodium citrate; 20-40 mM sodium acetate; and 20-30 mMsodium phosphate.
 2. The synthetic platelet storage media of claim 1wherein said media has a pH of approximately 7.2 and an osmolarity ofapproximately 300 mOsm/Kg.
 3. The synthetic platelet storage media, ofclaim 1 wherein said sodium phosphate comprises NaH₂PO₄ and Na₂HPO₄.